Optimized and Simplified Technique for the Production and Culture of Precision-Cut Liver Slices

Summary

A protocol for the production and culture of Precision-cut Liver Slices (PCLS) for the study of mouse livers. The article focuses on key aspects of the protocol, which only requires standard laboratory equipment with access to a vibratome and allows survival of PCLS for a minimum of 4 days.

Abstract

This protocol presents a simple system for the creation and culture of Precision-cut Liver Slices (PCLS). PCLS contains all cells in an intact environment and, therefore, resembles a mini model of the whole organ. They enable the study of live tissues while replicating their complex phenotypes. This protocol allows the preparation of slices from mouse livers using a vibratome and standard laboratory equipment. Protocols for producing and culturing PCLS lack standardization and can vary quite drastically depending on the tissue of interest, the type of vibratome used, and the need for oxygen. These can be difficult to reproduce in some laboratories that have only access to a basic vibratome and common tissue culture facilities. We have put together a protocol focusing on the importance of some key steps within the varied protocols already available. This protocol, therefore, emphasizes the importance of the embedding method, the cutting orientation, a dynamic versus a static system, and the relevance of a minimum volume of culture. This protocol can be established and reproduced in a simple manner in most laboratories that have access to a basic tissue slicer. Taken together and following this protocol, PCLS can stay alive for a minimum of 4 days. PCLS is a simple, economical, and reproducible model to study pathophysiological and therapeutic screening for organs such as the liver.

Introduction

Precision-cut tissue slices (PCTS) are thin sections of organs. They allow the preservation of the architecture of the organ replicating a mini-organ while preserving the 3-dimensional aspect of neighboring cells and extracellular matrix. It is an appealing model due to its easy access, cost-saving, and less labor-intensive characteristics while preserving the tissue architecture.

PCTS fill a gap between in vitro cell studies and in vivo animal research, overcoming most disadvantages of both models. PCTS has been generated from various organs, such as the liver¹, intestines²

Protocol

Wild-type CD57Bl/6J mice were purchased from Charles River Laboratories. Mice had free access to food and water, housed in individually ventilated cages with controlled temperature and humidity conditions and with a 12 h light cycle. Animals aged 3 weeks were sacrificed, and livers were promptly harvested without perfusion. All animal work was approved following local ethical review by the University College London Animal Welfare and Ethical Review Board and performed under Home Office project license PP9223137 and in ac.......

Discussion

We demonstrate that producing and culturing PCLS can be easily achieved while ensuring a half-life of at least 4 days. This protocol recapitulates five critical steps: the embedding method if this type of vibratome is used, the orientation of cutting, a dynamic system of culture, a minimal volume of culture, and the use of inserts.

Protocols for the production and culture of PCLS are commonly available. However, they do lack standardization; they might focus on similar and specific points of t.......

Materials

Name	Company	Catalog Number	Comments
3 cm petri dish	Any		any suitable for cell culture
6, 12, 24 well culture plate	Any		any suitable for cell culture
Cyanoacrylate super glue	Any
D-Glucose	Gibco	A24940
Eosin	Merck	HT110316
Ethanol	Any
Fetal Bovine serum	ThermoFisher	26400044
Gentamycin	Gibco	15750060
Hematoxylin	Merck	51275
HEPES	Gibco	H0887
inserts 8um, for 12 well plates	Strastedt	83.3931.800
inserts 8um, for 24 well plates	Strastedt	83.3932.800
inserts 8um, for 6 well plates	Strastedt	83.3930.800
KREBS	Merck	K3753
Laminar Flow Hood			Hepa air filtration
Low melting agarose	ThermoFisher	16520050
MTS tetrazolium reagent	Abcam	ab197010
multi-well plate reader	Any
PBS tablets	ThermoFisher	P4417
Penicillin/Streptomycin	Gibco	15140-122
Scalpel blade	Any
Surgical forceps	Any		with a flat square-tip
Surgical scissors	Any
Vibratome	Leica	VT1000 S
William’s Medium E with GlutaMAX (WME)	ThermoFisher	W4125